FRK cancer-related mutations: Effect on enzymatic activity and cellular processes

Cancer-associated FRK mutations have not been thoroughly studied however, a previous study analyzed six cancer related mutations of BRK L16F, R131L, V253M, N317S, L343F, P450L; and it was found that there are similar residues in FRK. We identified R64P, K87N, S145R, K265R, N359I, del V378-F379 (VF) found in breast cancer, large intestine cancer, ovarian cancer, and liver cancer. This study analyzes the effect of the mutation of these similar residues in the BRK study, as well as uncharacterized cancer related mutations of FRK. Mutating two of the similar residues maintained the same effects in BRK and FRK whereas the remaining mutants in FRK had no effect on activity. The uncharacterized cancer related mutations of FRK produced interesting results where the mutations in kinase domain (K265R, N359I, del VF) reduced, inactivated and increased kinase activity respectively. Proliferation, migration and invasion assays were also performed with the R64P, K265R, N358I and VF mutations. The proliferation data revealed that the cell line expressing wild type FRK reduced cell growth which is consistent with past findings. The other mutations also resulted in reduced cell growth in MDA-MB-231 breast cancer cells but were not significantly different to wild type FRK. The invasion assay results show that the wild type FRK significantly reduced invasion compared to the MDA-MB-231 cells compared to parental controls. Differing from the proliferation assay, the mutations R64P, K265R, N359I, and VF showed no reduction in cell invasiveness compared to the parental. The mutations expressed transiently in HEK293 were found to only impact STAT3 phosphorylation where all FRK constructs induced STAT3 phosphorylation except for N359I. Binding experiments were performed using rotational anisotropy and four peptides derived from FRK C terminus (ETDSS(pY)SDANN), SH2 consensus binding sequence (HF(pY)ENI), PTEN (PNVEEPSNPEASSS), and BRCA1 (DTYLIPQIPHSHY). The wild type SH2 domain was able of binding to both peptides from the FRK C terminus and the SH2 consensus binding sequence with a Kd of 2.5 M and 0.4 M respectively. The S145R SH2 domain was also capable of interacting with both peptides with a Kd of 1.4 M with the FRK C terminus and 0.8 M with the SH2 consensus binding sequence. Unfortunately, the SH3 domain was unable to bind to the sequences selected